Multi-User Equipment approved in grant 2018/04375-2: Flow citometer model BD Accuri C6TM plus system and Accuri C6 plus Dell Optiplex workstation bundle

• Grant number: 19/08411-6
• Support Opportunities: Multi-user Equipment Program
• Start date: June 01, 2019
• End date: May 31, 2026
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Maria Isabel Nogueira Cano
• Grantee: Maria Isabel Nogueira Cano
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated research grant: 18/04375-2 - Studies about the biogenesis and composition of the Leishmania spp. ribonucleoprotein complex and its regulation, AP.TEM
• EMU web page: Página do Equipamento Multiusuário não informada
• Type of equipment: Processos Biológicos - Caracterização - Contagem células (inclui citômetros)
• Manufacturer: Fabricante não informado
• Model: Modelo não informado

Abstract

Leishmaniasis are tropical diseases transmitted by primitive parasites of the genus Leishmania, family Trypanosomatidae. For these diseases, which can be present in different clinical forms, there are no efficient control and treatment. The actual treatment protocols are highly toxic and expensive and can lead to the resistance of parasites and vectors. Knowledge about the parasite molecular machineries is crucial for recognizing new anti-Leishmania therapeutic targets. Telomeres are of great interest since they play fundamental roles in genome maintenance and cell proliferation. Telomeres are the physical ends of the chromosomes maintained by the action of telomerase ribonucleoprotein (RNP) minimally composed by, TERT (Telomerase Reverse Transcriptase), and the long non coding RNA TER (Telomerase RNA), which contains the template sequence for telomere addition. The biogenesis and assemble of the telomerase RNP is well known in model organisms and is entirely due to a network of structural interactions between TERT, TER and accessory proteins. Once assemble, the telomerase RNP interacts dynamically with some telomeric proteins whose role is to recruit telomerase to telomere elongation. This interaction is strictly regulate and vital for telomere maintenance. We have already identified and partially characterized Leishmania spp. telomerase activity and the genes encoding both TERT and TER. However, we were not able to characterize the telomerase RNP complex, and to identify the proteins that act in its biogenesis and the partners that help in complex assemble and regulation. In this context, it is important to remind that our group has already described different components of parasite telomeric chromatin with emphasis to the proteins that bind double and single-strand telomeric DNA as well as the lncRNA TERRA, which in other eukaryotes can regulate telomerase activity and cell life span. The present work has the aim of: i) structurally characterize the interactions between TERT domains and the telomeric DNA using biochemical and biophysical approaches, ii) try to obtain the cristalographic structure of TERT domains, iii) to characterize the interactions between TERT and TER (LeishTER), iiiiv) to study the functional roles played by TERT e and TER in L. major and L. infantum telomere maintenance and parasite lifespan by inducing knockout (CRISPR), overexpression of TERT, TER and PINX1 and treating parasites with BIBR1532 (telomerase inhibitor), iv) to use proteomic analysis to compare the constitution of the telomerase RNP complex in L. major and L. infantum lineages with different genetic backgrounds (i.e. knockouts and overexpressors) e v) to study the role played by lncRNA TERRA in telomerase and telomere length regulation using transcriptomic analysis and TERRA knockout. Here is worth to mention that although the components of parasite telomeres share some structural or functional conservation with other eukaryotes, they present many features that are exclusive to the genus. In this way, to study the biogenesis of parasite telomerase RNP and its interactions with telomeric DNA, the telomeric chromatin and TERRA, may help us to understand telomere evolution and to disturb parasite homeostasis, and therefore to find new parasite-specific targets against leishmaniasis. (AU)